Sucrose phosphate synthase (SPS) catalyzes the transfer of glycosyl group of uridine diphosphate glucose (UDP-G) to fructose-6-phosphate (F6P) to form sucrose-6-phosphate (S6P). Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20 kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.

Studies into the cell death program termed apoptosis have resulted in new information regarding how cells control and execute their own demise, including insights into the mechanism by which death-preventing factors can inhibit Bax-induced caspase activation. We investigated high temperature stress-induced cell death in Brassica rapa. Using a yeast functional screening from a Brassica rapa cDNA library, the BH5-127 EST clone encoding an apoptotic suppressor peptide was identified. However, a phylogenic tree showed that BH5-127 clusters within a clade containing SUMO-1 (Small Ubiquitin-like Modifier- 1). BH5-127 was confirmed similar to have function to SUMO-1 as Fas suppression. Expression of BH5-127 showed that substantial suppression of cell death survived on SD-galactose-Leu--Ura- medium. The results suggest that BrSE (Brassica rapa Sentrin EST, BH5-127) is one of the important regulatory proteins in programming cell death, especially in the seedling stage of Chinese cabbage.

Genetic Transformation and Expression of Sucrose Phosphate Synthase Mutant in Tomato Plant ABSTRACTSucrose phosphate synthase (SPS) is a key enzyme responsible for sucrose biosynthesis. In its regulation, SPS activity is modulated by an allosteric effector glucose-6-phosphate (G6P) suggested to have an ability to bind SPS N-terminus domain. To understand the role of N-terminus in regulating SPS, the SPS gene was mutated with the deletion of N-terminus domain (∆N-SPS). The ∆N-SPS gen was transformed into tomato plants with 5% transformation efficiency. Three transgenic tomato plant 4.20, 5.5.1, and 5.10 were obtained and confirmed by PCR analysis. Transgenic tomato expression was characterized by enzymatic analysis. Result showed that the G6P allosteric regulation in transgenic ∆N-SPS had lost and the SPS activity increased by 2-fold compared to non-transgenic plant. This showed that N-terminus domain-deleted SPS could be actively expressed in plant. Keywords: enzyme, genetic transformation, N-terminus domain deletion, sucrose phosphate synthase, tomato ABSTRAKSucrose phosphate synthase (SPS) merupakan enzim kunci yang bertanggung jawab dalam sintesis sukrosa. Dalam regulasinya, aktifitas SPS dipengaruhi oleh alosterik efektor glukosa-6-fosfat (G6P) yang diduga dapat berikatan pada domain N-terminus SPS. Untuk mengetahui peran N-terminus pada regulasi SPS, dilakukan mutasi SPS dengan penghilangan domain N-terminus (∆N-SPS). Gen ∆N-SPS diinsersi pada tanaman tomat melalui transformasi genetik dengan efisiensi transformasi 5%. Tiga tanaman transgenik tomat (event4.20; 5.5.1; dan 5.10) didapatkan dan positif terkonfirmasi melalui analisis PCR. Ekspresi mutan dikarakterisasi melalui analisis enzimatik. Hasil menunjukkan bahwa tanaman tomat transgenik ∆N-SPS tidak dipengaruhi regulasi alosterik G6P dan aktifitas SPS 2 kali lipat lebih tinggi daripada tanaman bukan transgenik. Ini menunjukkan bahwa SPS dengan delesi domain N-terminus dapat terekspresi aktif pada tanaman. Kata Kunci: delesi domain N-terminus, enzim, sucrose phosphate synthase, tomat, transformasi genetik

Sugarcane mosaic virus (SCMV, genus Potyvirus, family Potyviridae) is a prominent pathogen of sugarcane (Saccharum sp. hybrids). It can cause losses in susceptible varieties, in crop as well as sugar production, economically. Although it has been studied in major sugar-producing countries, research on the definement of SCMV from Indonesian isolates based on molecular study has been very limited. This study aimed to obtain a proper recombinant antigens emanating from coat protein of SCMV from Indonesian isolate in order to produce polyclonal antibodies that cann be used for immunodiagnosis assays in a subsequent study. A gene-encoding coat protein of SCMV (CP-SCMV) was amplified using RT-PCR and cloned into vector pJET1.2. The cDNA was inserted into 6X His-tag expression plasmid of pET28a(+) and over-expressed in Escherichia coli BL21(DE3) to produce a recombinant protein. The highest expression was found in 0.1M IPTG induction media for 5 h at 37oC. SDS-PAGE analysis clarified that the recombinant CP-SCMV remained as an insoluble fraction. Purifications was carried out by the affinity Ni-NTA resin, followed by electroelution to obtain a highly purified protein. To meet the quality requirements of a proper antigen, the highly purified protein was concentrated. A molecular weight of the rCP-SCMV (approximately 40 kDa) was clearly observed by 10% SDS-PAGE at the concentration of 16.184 mg/mL.